Rbp4 antagonists for the treatment of age-related macular degeneration and stargardt disease

ABSTRACT

A method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure of any one of Formulas I-IV described herein, or a pharmaceutically acceptable salt thereof.

This application is a continuation of U.S. Ser. No. 14/775,552, filed Sep. 11, 2015, a §371 national stage of PCT International Application No. PCT/US2014/026523, filed Mar. 13, 2014, claiming the benefit of U.S. Provisional Application No. 61/785,227, filed Mar. 14, 2013, the contents of each of which are hereby incorporated by reference in their entirety.

This invention was made with government support under grant number NS067594, NS074476, EY019861, and EY012951 awarded by the National Institutes of Health. The government has certain rights in the invention.

Throughout this application, certain publications are referenced in parenthesis. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. It is estimated that 62.9 million individuals worldwide have the most prevalent atrophic (dry) form of AMD; 8 million of them are Americans. Due to increasing life expectancy and current demographics this number is expected to triple by 2020. There is currently no FDA-approved treatment for dry AMD. Given the lack of treatment and high prevalence, development of drugs for dry AMD is of upmost importance. Clinically, atrophic AMD represents a slowly progressing neurodegenerative disorder in which specialized neurons (rod and cone photoreceptors) die in the central part of the retina called macula (1). Histopathological and clinical imaging studies indicate that photoreceptor degeneration in dry AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath photoreceptors and provides critical metabolic support to these light-sensing neuronal cells. Experimental and clinical data indicate that excessive accumulation of cytotoxic autofluorescent lipid-protein-retinoid aggregates (lipofuscin) in the RPE is a major trigger of dry AMD (2-9). In addition to AMD, dramatic accumulation of lipofuscin is the hallmark of Stargardt Disease (STGD), an inherited form of juvenile-onset macular degeneration. The major cytotoxic component of RPE lipofuscin is pyridinium bisretinoid A2E (FIG. 1). Additional cytotoxic bisretinoids are isoA2E, atRAL di-PE, and A2-DHP-PE (40, 41). Formation of A2E and other lipofuscin bisretinoids, such as A2-DHP-PE (A2-dihydropyridine-phosphatidylethanolamine) and atRALdi-PE (all-trans-retinal dimer-phosphatidylethanolamine), begins in photoreceptor cells in a non-enzymatic manner and can be considered as a by-product of the properly functioning visual cycle.

A2E is a product of condensation of all-trans retinaldehyde with phosphatidyl-ethanolamine which occurs in the retina in a non-enzymatic manner and, as illustrated in FIG. 4, can be considered a by-product of a properly functioning visual cycle (10). Light-induced isomerization of 11-cis retinaldehyde to its all-trans form is the first step in a signaling cascade that mediates light perception. The visual cycle is a chain of biochemical reactions that regenerate visual pigment (11-cis retinaldehyde conjugated to opsin) following exposure to light.

As cytotoxic bisretinoids are formed during the course of a normally functioning visual cycle, partial pharmacological inhibition of the visual cycle may represent a treatment strategy for dry AMD and other disorders characterized by excessive accumulation of lipofuscin (25-27, 40, 41).

SUMMARY OF THE INVENTION

The present invention relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   ring A is benzene optionally further substituted;     -   R¹ is an optionally substituted branched C₃₋₆ alkyl group;     -   X¹ is an O, S, SO, SO₂ or NH;     -   X² is a bond or a C₁₋₃ alkylene group;     -   ring B is azetidine, pyrrolidine or piperidine;     -   X³ is CO or SO₂;     -   R² is a substituent, provided that     -   (1) when —X¹—X²— is —NH— and ring B is piperidine, then X³ is         CO;     -   (2) when X³ is CO, then R² is not a tert-butoxy group, or a salt         thereof,     -   or a pharmaceutically acceptable salt thereof.

The present invention also relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   ring A is a benzene ring optionally further substituted;     -   ring B is a piperazine ring optionally further substituted; and     -   R is a substitutent,     -   or a pharmaceutically acceptable salt thereof.

The present invention further relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   A is O, NH, or S;     -   B is a bond, —(C₂-C₇)alkyl, —(C₂-C₇)alkenyl, —(C₃-C₈)cycloalkyl,         —(C₂-C₇) heteroalkyl, —(C₃-C₈) heterocycloalkyl,         —(C₃-C₈)cycloalkenyl, —(C₃-C₈) heterocycloalkenyl;     -   D is isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl,         sec-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl,         methylenecyclopropyl, methylenecyclobutyl, methylenecyclopentyl;     -   E is (C═O)—OR, —O—(C═O)—R, —(C═O)—R, —OR, a carboxylic acid         bioisostere, —(C═O)—NR¹R, NR¹—(C═O)—R, —(C₁-C₇)alkyl-(C═O)—OR,         or —(C₁-C₇)alkyl-(C═O) NR¹R;     -   R is H or

-   -   G is OR¹, —(C₁-C₆)alkyl, —(C₁-C₆)alkyl-OR¹, halogen, —CO₂R¹,         —(C₁-C₆)alkyl-CO₂R¹, NHR¹, —(C₁-C₆)alkyl-NHR¹, —(C═O)NHR¹,         —(C₁-C₆)alkyl-(C═O)NHR¹, —NHR¹(C═O)R¹,         —(C₁-C₆)alkyl-NHR¹(C═O)R¹;     -   R¹ is H or —(C₁-C₆)alkyl;     -   X is a halogen;     -   or an active metabolite, or a pharmaceutically acceptable         prodrug, salt, or solvate thereof.

The present invention yet further relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   ring A is a 5-membered non-aromatic heterocycle optionally         further substituted by one substitutent;     -   ring B is an optionally further substituted benzene ring; and     -   X is a bond, O, CH₂O, OCH₂, CH₂, (CH₂)₂, S, CH₂S, SCH₂, S(O),         CH₂S(O), S(O)CH₂, S(O)₂, CH₂S(O)₂ OR S(O)₂CH₂, provided that

-   {(3S,5R)-1-[4-(trifluoromethyl)benzyl]-5-[4-(trifluoromethyl)phenyl]pyrro-lidin-3-yl}acetic     acid,

-   {(3S,5R)-1-[2,5-bis(trifluoromethyl)benzyl]-5-[4-(trifluoromethyl)phenyl]-pyrrolidin-3-yl}acetic     acid,

-   {4-oxo-3-[(3-(trifluoromethyl)phenyl]-1,3-thiazolidin-5-yl}acetic     acid,

-   {2-oxo-1-[3-(trifluoromethyl)phenyl]pyrrolidin-3-yl}acetic acid,

-   {3-[4-fluoro-3-(trifluoromethyl)phenyl]-4-oxo-1,3-oxazolidin-5-yl}acetic     acid,

-   {4-oxo-3-[3-(trifluoromethyl)phenyl]-1,3-oxazolidin-5-yl}acetic     acid,

-   {3-[2-chloro-5-(trifluoromethyl)phenyl]-4-oxo-1,3-thiazolidin-5-yl}acetic     acid, and

-   {5-oxo-1-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1H-pyrazol-3-yl}acetic     acid are excluded,     -   or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure of bisretinoid A2E, a cytotoxic component of retinal lipofuscin.

FIG. 2. Structure of bisretinoid atRAL di-PE (all-transretinal dimer-phosphatidyl ethanolamine), a cytotoxiccomponent of retinal lipofuscin. R1 and R2 refer to various fatty acid constituents.

FIG. 3. Structure of bisretinoid A2-DHP-PE, a cytotoxic component of retinal lipofuscin.

FIG. 4. Visual cycle and biosynthesis of A2E. A2E biosynthesis begins when a portion of all-trans-retinal escapes the visual cycle (yellow box) and non-enzymatically reacts with phosphatidyl-ethanolamine forming the A2E precursor, A2-PE. Uptake of serum retinol to the RPE (gray box) fuels the cycle.

FIG. 5. Three-dimensional structure of the RBP4-TTR-retinol complex. Tetrameic TTR is shown in blue, light blue, green and yellow (large boxed region). RBP is shown in red (unboxed region) and retinol is shown in gray (small boxed region) (28).

FIG. 6. Structure of fenretinide, [N-(4-hydroxy-phenyl)retinamide, 4HRP], a retinoid RBP4 antagonist.

FIG. 7. Schematic depiction of the HTRF-based assay format for characterization of RBP4 antagonists disrupting retinol-induced RBP4-TTR interaction.

FIG. 8. Dose titrations of all-trans retinol (panels A and B, blue), Compound 1 (red, A), and fenretinide (red, B) in the HTRF-based RBP4-TTR interaction assay.

FIG. 9. Dose titrations of Compound 1 and fenretinide in the presence of all-trans retinol in the HTRF-based RBP4-TTR interaction assay.

FIG. 10. Compound 1 does not reduce ERG b-wave after photobleaching.

FIG. 11. Reduction in serum RBP4 in response to Compound 1 treatment. Effect of long-term oral A1120 administration on serum RBP4 in Abca4−/− mice. Serum RBP4 levels were measured with ELISA test in vehicle-treated wild-type mice (green columns), vehicle-treated Abca4−/− mice (blue columns), and A1120-treated Abca4−/− mice (red columns) at indicated timepoints. A1120 formulated in a chow was dosed at 30 mg/kg. Compared with Day 0, statistically significant 64% RBP4 reduction at Week 3 and 75% RBP4 reduction at Week 6 is seen in the A1120 treatment group (p<0.05). Changes in RBP4 levels at different timepoints within the vehicle-treated wild-type and vehicle-treated Abca4−/− groups were not statistically significant.

FIG. 12. Reduction of toxin bisretinoids by Compound 1.

FIG. 13. Effect of A1120 treatment on the levels of lipofuscin fluorophores in eyes of the Abca4−/− mice. Bisretinoids were extracted from the eyecups of vehicle-treated wild-type mice, vehicle-treated Abca4−/− mice, and A1120-treated Abca4−/− mice after 6 weeks of dosing and analyzed by HPLC. 13A: The representative reverse phase HPLC chromatogram (monitoring at 430 nm) of an extract from eyecups of A1120-treated Abca4−/− mice. Insets on the top show UV-visible absorbance spectra of A2E and iso-A2E. 13B: Chromatographic monitoring at 510 nm, retention time 40-50 minutes, for A2-DHP-PE (A2-dihydropyridine-phosphatidylethanolamine) and atRALdi-PE (all-transretinal dimmer-phosphatidylethanolamine) detection with insets on the top showing absorbance UV-visible spectra of A2-DHP-PE and atRALdi-PE. 13C: Levels of A2E, A2-DHP-PE and atRALdi-PE in vehicle-treated wild-type mice, vehicle-treated Abca4−/− mice, and A1120-treated Abca4−/− mice after 6 weeks of dosing showing 45-50% reduction in bisretinoid levels in response to A1120 treatment.

FIG. 14A: Analysis of Compound 1 in SPA-based RBP4 binding assay. Titration was conducted 7 times. IC₅₀ values calculated in seven experiments were 0.00579, 0.0229, 0.0148, 0.0138, 0.0126, 0.0156 and 0.00901 (in μM).

FIG. 14B: Analysis of Compound 1 in HTRF-based retinol-dependent RBP4-TTR interaction assay. Titration was conducted 9 times. IC₅₀ values calculated in nine experiments were 0.182, 0.119, 0.195, 0.139, 0.101, 0.109, 0.0848, 0.126 and 0.134 (in μM).

FIG. 14C: Analysis of Compound 64 in SPA-based RBP4 binding assay. IC₅₀ value calculated in this experiment was 0.0498 μM.

FIG. 14D: Analysis of Compound 64 in HTRF-based retinol-dependent RBP4-TTR interaction assay. IC₅₀ value calculated in this experiment was 1.27 μM.

FIG. 14E: Analysis of Compound 65 in SPA-based RBP4 binding assay. IC₅₀ value calculated in this experiment was 0.0199 μM.

FIG. 14F: Analysis of Compound 65 in HTRF-based retinol-dependent RBP4-TTR interaction assay. IC₅₀ value calculated in this experiment was 0.199 μM.

FIG. 14G: Analysis of Compound 48 in SPA-based RBP4 binding assay. IC₅₀ value calculated in this experiment was 0.00568 μM.

FIG. 14H: Analysis of Compound 48 in HTRF-based retinol-dependent RBP4-TTR interaction assay. IC₅₀ value calculated in this experiment was 0.106 μM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   ring A is benzene optionally further substituted;     -   R¹ is an optionally substituted branched C₃₋₆ alkyl group;     -   X¹ is an O, S, SO, SO₂ or NH;     -   X² is a bond or a C₁₋₃ alkylene group;     -   ring B is azetidine, pyrrolidine or piperidine;     -   X³ is CO or SO₂;     -   R² is a substituent, provided that     -   (3) when —X¹—X²— is —NH— and ring B is piperidine, then X³ is         CO;     -   (4) when X³ is CO, then R² is not a tert-butoxy group, or a salt         thereof,     -   or a pharmaceutically acceptable salt thereof.

Specific examples of compounds having Formula (I) are described in, e.g., U.S. Patent Application Publication No. US 2010/0292206 A1, published on Nov. 18, 2010, the entire content of which is hereby incorporated by reference herein.

The present invention also relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   ring A is a benzene ring optionally further substituted;     -   ring B is a piperazine ring optionally further substituted; and     -   R is a substitutent,     -   or a pharmaceutically acceptable salt thereof.

Specific examples of compounds having Formula (II) are described in, e.g., PCT International Application Publication No. WO 2010/119992 A1, published on Oct. 21, 2010, the entire content of which is hereby incorporated by reference herein.

The present invention further relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   A is O, NH, or S;     -   B is a bond, —(C₂-C₇)alkyl, —(C₂-C₇)alkenyl, —(C₃-C₈)cycloalkyl,         —(C₂-C₇) heteroalkyl, —(C₃-C₈) heterocycloalkyl,         —(C₃-C₅)cycloalkenyl, —(C₃-C₈)heterocycloalkenyl;     -   D is isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl,         sec-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl,         methylenecyclopropyl, methylenecyclobutyl, methylenecyclopentyl;     -   E is (C═O)—OR, —O—(C═O)—R, —(C═O)—R, —OR, a carboxylic acid         bioisostere, —(C═O)—NR¹R, NR¹— (C═O)—R, —(C₁-C₇)alkyl-(C═O)—OR,         or —(C₁-C₇)alkyl-(C═O) NR¹R;     -   R is H or

-   -   G is OR¹, —(C₁-C₆)alkyl, —(C₁-C₆)alkyl-OR¹, halogen, —CO₂R¹,         —(C₁-C₆)alkyl-CO₂R¹, NHR¹, —(C₁-C₆)alkyl-NHR¹, —(C═O)NHR¹,         —(C₁-C₆)alkyl-(C═O)NHR¹, —NHR¹(C═O)R¹, —(C₁-C₆)alkyl-NHR¹ (C═O)         R¹;     -   R¹ is H or —(C₁-C₆)alkyl;     -   X is a halogen;     -   or an active metabolite, or a pharmaceutically acceptable         prodrug, salt, or solvate thereof.

In one aspect of method the compound of Formula (III) has the following structure:

-   -   wherein     -   A is O, NH, or S;     -   B is a bond, —(C₂-C₇)alkyl, —(C₂-C₇)alkenyl, —(C₃-C₈)cycloalkyl,         —(C₂-C₇) heteroalkyl, —(C₃-C₈) heterocycloalkyl,         —(C₃-C₈)cycloalkenyl, —(C₃-C₈)heterocycloalkenyl;     -   E is (C═O)—OR, —O—(C═O)—R, —(C═O)—R, —OR, a carboxylic acid         bioisostere, —(C═O)—NR¹R, NR¹—(C═O)—R, —(C₁-C₇)alkyl-(C═O)—OR,         or —(C₁-C₇)alkyl-(C═O) NR¹R;     -   R is H or

-   -   G is OR¹, —(C₁-C₆)alkyl, —(C₁-C₆)alkyl-OR¹, halogen, —CO₂R¹,         —(C₁-C₆)alkyl-CO₂R¹, NHR¹, —(C₁-C₆)alkyl-NHR¹, —(C═O)NHR¹,         —(C₁-C₆)alkyl-(C═O)NHR¹, —NHR¹(C═O)R¹, —(C₁-C₆)alkyl-NHR¹(C═O)         R¹;     -   R¹ is H or —(C₁-C₆)alkyl;     -   or an active metabolite, or a pharmaceutically acceptable         prodrug, salt, or solvate thereof.

Specific examples of compounds having Formula (III) are described in, e.g., PCT International Application Publication No. WO 2009/042444 A2, published on Apr. 2, 2009, the entire content of which is hereby incorporated by reference herein.

The present invention yet further relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

-   -   wherein     -   ring A is a 5-membered non-aromatic heterocycle optionally         further substituted by one substitutent;     -   ring B is an optionally further substituted benzene ring; and     -   X is a bond, O, CH₂O, OCH₂, CH₂, (CH₂), S, CH₂S, SCH₂, S(O),         CH₂S(O), S(O)CH₂, S(O)₂, CH₂S(O)₂ OR S(O)₂CH₂, provided that

-   {(3S,5R)-1-[4-(trifluoromethyl)benzyl]-5-[4-(trifluoromethyl)phenyl]pyrro-lidin-3-yl}acetic     acid,

-   {(3S,5R)-1-[2,5-bis(trifluoromethyl)benzyl]-5-[4-(trifluoromethyl)phenyl]-pyrrolidin-3-yl}acetic     acid,

-   {4-oxo-3-[(3-(trifluoromethyl)phenyl]-1,3-thiazolidin-5-yl}acetic     acid,

-   {2-oxo-1-[3-(trifluoromethyl)phenyl]pyrrolidin-3-yl}acetic acid,

-   {3-[4-fluoro-3-(trifluoromethyl)phenyl]-4-oxo-1,3-oxazolidin-5-yl}acetic     acid,

-   {4-oxo-3-[3-(trifluoromethyl)phenyl]-1,3-oxazolidin-5-yl}acetic     acid,

-   {3-[2-chloro-5-(trifluoromethyl)phenyl]-4-oxo-1,3-thiazolidin-5-yl}acetic     acid, and

-   {5-oxo-1-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1H-pyrazol-3-yl}acetic     acid are excluded,     -   or a pharmaceutically acceptable salt thereof.

Specific examples of compounds having Formula (IV) are described in, e.g., U.S. Patent Application Publication No. US 2011/0251187 A1, published on Oct. 13, 2011, the entire content of which is hereby incorporated by reference herein.

In some embodiments, the disease is further characterized by bisretinoid-mediated macular degeneration.

In some embodiments, the amount of the compound of the present method is effective to lower the serum concentration of RBP4 in the mammal.

In some embodiments, the amount of the compound of the present method is effective to lower the retinal concentration of a bisretinoid in lipofuscin in the mammal.

In some embodiments of the invention, the amount of the compound of the present method may be effective to lower the retinal concentration of a bisretinoid in lipofuscin in the mammal. In some embodiments, the bisretinoid is A2E. In some embodiments the bisretinoid is isoA2E. In some embodiments the bisretinoid is A2-DHP-PE. In some embodiments the bisretinoid is atRAL di-PE.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina may be Age-Related Macular Degeneration or Stargardt Disease.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is dry (atrophic) Age-Related Macular Degeneration.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Stargardt Disease.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Best disease.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is adult vitelliform maculopathy.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Stargardt-like macular dystrophy.

In some embodiments, bisretinoid-mediated macular degeneration may be Age-Related Macular Degeneration or Stargardt Disease.

In some embodiments, the bisretinoid-mediated macular degeneration is Age-Related Macular Degeneration.

In some embodiments, the bisretinoid-mediated macular degeneration is dry (atrophic) Age-Related Macular Degeneration.

In some embodiments, the the bisretinoid-mediated macular degeneration is Stargardt Disease.

In some embodiments, the bisretinoid-mediated macular degeneration is Best disease.

In some embodiments, the bisretinoid-mediated macular degeneration is adult vitelliform maculopathy.

In some embodiments, the bisretinoid-mediated macular degeneration is Stargardt-like macular dystrophy.

The bisretinoid-mediated macular degeneration may comprise the accumulation of lipofuscin deposits in the retinal pigment epithelium.

As used herein, “bisretinoid lipofuscin” is lipofuscin containing a cytotoxic bisretinoid. Cytotoxic bisretinoids include but are not necessarily limited to A2E, isoA2E, atRAL di-PE, and A2-DHP-PE (FIG. 1-3).

As used herein, the description “pharmaceutically active” is used to characterize a substance, compound, or composition suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; Nov. 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department of Health and Human Services, 30^(th) edition, 2010), which are hereby incorporated by reference. Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.

The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compound which has been modified by making acid or base salts of the compounds. In the case of the use of the compounds for treatment of bisretinoid-mediated macular degeneration, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines. The term “pharmaceutically acceptable salt” in this respect, refers to the relatively non-toxic, inorganic and organic base addition salts of the compounds. These salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting purified compounds in their free acid form with a suitable organic or inorganic base, and isolating the salt thus formed.

As used herein, “treating” means slowing, stopping, or preventing the progression of a disease. An embodiment of “treating bisretinoid-mediated macular degeneration” is delaying or preventing the onset, progression, or mitigating severity of vision loss.

The compounds used in the method of the present invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the mammal in need of the drug is treated or given another drug for the disease in conjunction with the compounds used in the method of the present invention. This combination therapy can be sequential therapy where the mammal is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

As used herein, a “pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the mammal. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier.

The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of the compound and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds used in the method of the present invention may comprise the compound alone, or mixtures of the compound with additional compounds used to treat lipofuscin-mediated macular degeneration. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.

The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the eye, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

The compounds used in the method of the present invention can be administered in a mixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297, issued Sep. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds used in the method of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.

The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxy-ethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, The compounds used in the method of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

The compounds used in the method of the present invention can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parentally, in sterile liquid dosage forms.

Gelatin capsules may contain the compounds used in the method of the present invention and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

For oral administration in liquid dosage form, the compounds used in the method of the present invention may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

The compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

The compounds used in the method of the present invention and compositions thereof of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.

The compounds and compositions of the present invention are useful for the prevention and treatment of lipofuscin-mediated macular degeneration.

Except where otherwise specified, when the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in “Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as ¹²C, ¹³C, or ¹⁴C. Furthermore, any compounds containing ¹³C or ¹⁴C may specifically have the structure of any of the compounds disclosed herein.

The compounds used in the method of the present invention may be prepared by techniques well know in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

The compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J. Hannaford, P. W. G. Smith, (Prentice Hall) 5^(th) Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5^(th) Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

As used herein, “alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted. Thus, C₁-C_(n) as in “C₁-C_(n) alkyl” is defined to include groups having 1, 2, . . . , n−1 or n carbons in a linear or branched arrangement. For example, C₁-C₆, as in “C₁-C₆alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, hexyl, and octyl.

It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as ¹H, ²H, or ³H. Furthermore, any compounds containing ²H or ³H may specifically have the structure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

This invention will be better understood by reference to the Examples which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

EXAMPLES Example 1. Synthesis of Compound 1

The compound 2(4-(2-(trifluoromethyl)phenyl)piperidine-1-carboxamido)benzoic acid has the structure:

termed “Compound 1” herein, and was obtained from Sigma (Sigma-Aldrich Corp., St. Louise Mo., USA, Catalogue No. A3111). Compound 1 is described in PCT/US2011/061763, the contents of which are hereby incorporated by reference.

Compound 1, has also been called A1120 and may be made by the following techniques described in Motani et al., 2009 as follows: A solution of methyl 2-isocyanatobenzoate (10.00 g, 56.4 mmol) in tetrahydrofuran (30 ml) was slowly added to a solution of 4-(2-(trifluoromethyl)phenyl)piperidine hydrochloride (14.3 g, 53.8 mmol, Sigma) and triethylamine 99% (8.99 ml, 64.5 mmol) in tetrahydrofuran (120 ml) at 0° C. The mixture was removed from the cooling bath and stirred at room temperature for 15 min, at which time LC/MS analysis indicated that the reaction was complete. EtOH (75 ml) and aqueous LiOH (2N, 95 ml) were then added, and the solution was stirred for 6 h at room temperature. Subsequently, aqueous HCl (2N, 150 ml) was added, and the resulting mixture was extracted with EtOAc (2×600 ml). The EtOAc extract was dried over MgSO4 and concentrated to an off-white solid. Recrystallization from EtOAc yielded 14.0 g (66%) of 2-(4-(2-(trifluoromethyl)phenyl)piperidine-1-carboxamido) benzoic acid as a white solid, which was homogeneous by analytical high-performance liquid chromatography (>99%).

Example 2. TR-FRZT Assay for Antagonists of Retinol-Induced RBP4-TTR Interaction

TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) is an assay format that can be used in characterization of compounds affecting protein-protein interactions (31-33). The HTRF (Homogeneous Time-Resolved Fluorescence) variant of TR-FRET is the most advanced as it has improved light capturing due to the use of Eu3+ cryptates. In the presence of retinol, RBP4-TTR interaction induces FRET that can be registered as increased ratio of 668/620 fluorescence signals. Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (FIG. 7).

The assay was developed using E. coli-expressed MBP-tagged RBP4 and commercially available TTR labeled directly with Eu3+ cryptate. In addition to MBP-RBP4 and Eu3+ (K)-TTR, a detector reagent anti-MBP-d2 was present in the mix. The assay was first optimized in the agonist mode; sensitivity and dynamic range of the assay was first mode in respect to RBP4, TTR and detection reagent concentrations. In order to determine the optimum concentration of all-trans retinol stimulating the RBP4-TTR interaction eight-point titration retinol titrations were performed along with titrations of Compound 1 and fenretinide (FIG. 8). It was demonstrated that all-trans retinol stimulates RBP4-TTR interaction in a dose dependent manner (FIG. 8) with EC₅₀ of ˜1.2 M. As expected, RBP4 antagonists Compound 1 and fenretinide did not induce RBP4-TTR interaction (FIG. 8).

Given that retinol is present in serum at micromolar concentrations and taking into account the results of retinol titrations, the assay was converted to the antagonist mode by testing fixed concentration of retinol within the 1-10 μM range and using the saturating 40 μM concentration of antagonists (fenretinide and Compound 1). The optimum retinol concentration in the antagonist mode in regard of assay sensitivity and dynamic range was found to be in the 4.5-6.5 μM range. Titrations of Compound 1 and fenretinide were conducted in the presence of retinol in order to characterize our starting compounds in the primary assay and prove that the assay is suitable for characterization of RBP4 antagonists (FIG. 9).

The two compounds, Compound 1 and fenretinide, antagonized the retinol-induced RBP4-TTR interaction with EC₅₀'s in the μM range (2.2 μM for Compound 1 and 17.3 μM for fenretinide).

Example 3. Compound 1 Efficacy in a Maamalian Model

The effectiveness of Compound 1 was tested in wild-type and Abca4−/− mice. The Abca4−/− mouse model manifests accelerated accumulation of lipofuscin in the RPE and is considered a pre-clinical efficacy model for a drug reducing lipofuscin accumulation. Compound 1 was orally dosed for 3 weeks at 30 mg/kg. There was approximately a 70% reduction in the serum RBP4 level in treated animals (FIG. 11). Additionally, it was discovered that that the levels of A2E/isoA2E and other bisretinoids were reduced by approximately 50% in treated mice (FIG. 12). The levels of A2-DHP-PE and atRAL di-PE were also reduced. These preclinical efficacy data show that Compound 1 is a potential small molecule treatment for dry AMD and Stargardt's disease.

Tissue Extraction and HPLC Analysis of Bisretinoids

Abca4/Abcr null mutant mice (albino) homozygous for Rpe65-Leu450 are bred genotyped and housed. Posterior eyecups of mice and RPE/choroids harvested from human donor eyes (National Disease Research Interchange, Philadelphia Pa.) are homogenized in phosphate buffered saline (PBS) using a glass tissue grinder and extracted in chloroform/methanol (2:1). Extracts are subsequently filtered through cotton and passed through a reverse phase cartridge (C8 Sep-Pak, Millipore) with 0.1% TFA (Aldrich Chemical Company, Milwaukee, Wis.) in methanol. After evaporation of solvent under argon gas, the extract is dissolved in 50% methanolic chloroform containing 0.1% TFA. An Alliance system (Waters, Corp, Milford, Mass.) equipped with 2695 Separation Module, 2996 Photodiode Array Detector, a 2475 Multi λ Fluorescence Detector and operating with Empower® software is used for HPLC analysis. An Atlantis® dC18 column (3 μm, 4.6×150 mm, Waters, USA) and a Delta Pak® C4 column (5 μm, 3.9×150 mm, Waters, USA) are employed. Gradients of water and acetonitrile (Fisher, Fair Lawn, N.J.) with 0.1% of TFA are used for mobile phase; details are provided in figure legends. HPLC quantification is carried out using the Empower® software to determine peak areas. Detection by photodiode array is set at 430 and 490 nm. Molar quantity per murine eye is determined using calibration curves constructed from known concentrations of purified external standards and by normalizing to the ratio of the HPLC injection volume (10 μL) versus total extract volume.

Example 4. TR-FRET Assay for Retinol-Induced RBP4-TTR Interaction

Bacterially expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP)-tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3)pLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5-ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu³⁺ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 μl per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7 nM of anti-MBP antibody conjugated with d2 (Cisbio). Titration of test compounds in this assay was conducted in the presence of 1 μM retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4° C. wrapped in aluminum foil. TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device). Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds). The TR-FRET signal was expressed as the ratio of fluorescence intensity: Flu₆₆₅/Flu₆₂₀×10,000.

Example 5. Scintillation Proximity RBP4 Binding Assay

Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations.

Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 μl per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM ³H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 μM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company).

Example 6. Animal Studies

Ten week-old Abca4 null mutant mice (129/SV×C57BL/6J) bred as previously described were used in the study. Abca4−/− (knockout) and Abca4+/+ (wild-type) mice were raised under 12 h on-off cyclic lighting with an in-cage illuminance of 30-50 lux. For long-term oral dosing A1120 was formulated into Purina 5035 rodent chow at Research Diets, Inc. (New Brunswick, N.J.) to ensure consistent 30 mg/kg daily oral dosing. Animals were administered the A1120-containing chow for 6 weeks.

Example 7. Serum RBP4 Measurements

Blood samples were collected from a tail vein at days 0, 21 and 42 of the A1120 dosing. Whole blood was drawn into a centrifuge tube and was let clot at room temperature for 30 min followed by centrifugation at 2,000×g for 15 minutes at +4′C to collect serum. Serum RBP4 was measured using the RBP4 (mouse/rat) dual ELISA kit (Enzo Life Sciences) following the manufacturer's instructions.

Example 8. Biretinoid Extraction and Analysis

Following euthanasia, posterior eye cups were pooled and homogenized in PBS using a tissue grinder. An equal volume of a mixture of chloroform and methanol (2:1) was added, and the sample was extracted three times. To remove insoluble material, extracts were filtered through cotton and passed through a reverse phase (C18 Sep-Pak, Millipore) cartridge with 0.1% TFA in methanol. After the solvent had been removed by evaporation under argon gas, the extract was dissolved in methanol containing 0.1% TFA, for HPLC analysis. For quantification of bisretinoids of RPE lipofuscin, a Waters Alliance 2695 HPLC system was employed with an Atlantis dC18 column (Waters, 4.6 mm×150 mm, 3 μm) and the following gradient of acetonitrile in water (containing 0.1% trifluoroacetic acid): 90 to 100% from 0 to 10 min and 100% acetonitrile from 10 to 20 min, with a flow rate of 0.8 mL/min with monitoring at 430 nm. The injection volume was 10 μL. Extraction and injection for HPLC were performed under dim red light. Levels of bisretinoid were determined by reference to external standards of HPLC-purified compound.

Example 9. Compounds of Formulas I-IV

Compounds having the structure of any one of Formulas I-IV used in the method of the present invention function analogously to Compound 1. Synthesis of these compounds are described in, e.g., U.S. Patent Application Publication No. US 2010/0292206 A1, PCT International Application Publication No. WO 2010/119992 A1, PCT International Application Publication No. WO 2009/042444 A2, and U.S. Patent Application Publication No. US 2011/0251187 A1, the entire content of each of which is hereby incorporated by reference herein.

Further, those having ordinary skill in the art of organic synthesis will appreciate that modifications to general procedures described herein and synthetic routes contained in this application can be used to synthesize compounds used in the method of the present invention. Suitable organic transformations are described in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (Wiley-Interscience; 6^(th) edition, 2007), the entire content of which is hereby incorporated by reference.

Example 10. Correlation Between A1120-Induced Serum RBP4 Reduction and Inhibition of Bisretinoid Accumulation in the Retina

To determine whether A1120 has an effect on retinal production of lipofuscin fluorophores we administered the compound at the daily 30 mg/kg dose to Abca4−/− mice for a period of 6 weeks. Blood samples collected from the treatment and control groups at baseline, Day 21 and Day 42 were used to measure serum RBP4 in order to correlate RBP4 levels with reduction in formation of lipofuscin bisretinoids. As shown in FIG. 13, chronic oral administration of A1120 at 30 mg/kg to Abca4−/− mice induced a 64% decrease in serum RBP4 level at Day 21 and a 75% decrease at Day 42. Levels of lipofuscin fluorophores (A2E, A2-DHP-PE and all-trans-retinal dimer-PE) were determined at the end of the 42-day treatment period using quantitative HPLC.

Representative chromatogram of lipofuscin fluorophores from eyecups of vehicle-treated Abca4−/− mice along with absorbance spectra for the indicated peaks is shown in FIG. 14, A and B. As shown in FIG. 14, C the levels of bisretinoid accumulation were 3-4 times higher in the vehicle-treated Abca4−/− mice than in wild-type controls. Administration of A1120 reduces the production of A2E, A2-DHP-PE, and atRAL di-PE in A1120-treated Abca4−/− mice in comparison to the vehicle-treated Abca4−/− animals by approximately 50%. This result clearly demonstrated that A1120 can inhibit in vivo accumulation of toxic lipofuscin bisretinoids in the animal model of enhanced lipofuscinogenesis. We did not note any obvious signs of compound toxicity such as weight loss or reduction in food consumption during the 6 week-long chronic A1120 dosing.

Example 11. Administration of a Compound of Formula I

An amount of a compound of Formula I as described herein is administered to the eye of a subject afflicted with AMD. The amount of the compound is effective to treat the subject.

An amount of a compound of Formula I as described herein is administered to the eye of a subject afflicted with Stargardt disease. The amount of the compound is effective to treat the subject.

Example 12. Administration of a Compound of Formula II

An amount of a compound of Formula II as described herein is administered to the eye of a subject afflicted with AMD. The amount of the compound is effective to treat the subject.

An amount of a compound of Formula II as described herein is administered to the eye of a subject afflicted with Stargardt disease. The amount of the compound is effective to treat the subject.

Example 13. Administration of a Compound of Formula III

An amount of a compound of Formula III as described herein is administered to the eye of a subject afflicted with AMD. The amount of the compound is effective to treat the subject.

An amount of a compound of Formula III as described herein is administered to the eye of a subject afflicted with Stargardt disease. The amount of the compound is effective to treat the subject.

Example 14. Administration of a Compound of Formula IV

An amount of a compound of Formula IV as described herein is administered to the eye of a subject afflicted with AMD. The amount of the compound is effective to treat the subject.

An amount of a compound of Formula Iv as described herein is administered to the eye of a subject afflicted with Stargardt disease. The amount of the compound is effective to treat the subject.

DISCUSSION

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Its prevalence is higher than that of Alzheimer's disease. There is no treatment for the most common dry form of AMD. Dry AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and provides critical metabolic support to these light-sensing cells. RPE dysfunction induces secondary degeneration of photoreceptors in the central part of the retina called the macula. Experimental data indicate that high levels of lipofuscin induce degeneration of RPE and the adjacent photoreceptors in atrophic AMD retinas. In addition to AMD, dramatic accumulation of lipofuscin is the hallmark of Stargardt's disease (STGD), an inherited form of juvenile onset macular degeneration. The major cytotoxic component of RPE lipofuscin is a pyridinium bisretinoid A2E. A2E formation occurs in the retina in a non-enzymatic manner and can be considered a by-product of a properly functioning visual cycle. Given the established cytotoxic affects of A2E on RPE and photoreceptors, inhibition of A2E formation could lead to delay in visual loss in patients with dry AMD and STGD. It was suggested that small molecule visual cycle inhibitors may reduce the formation of A2E in the retina and prolong RPE and photoreceptor survival in patients with dry AMD and STGD. Rates of the visual cycle and A2E production in the retina depend on the influx of all-trans retinol from serum to the RPE. RPE retinol uptake depends on serum retinol concentrations. Pharmacological downregulation of serum retinol is a valid treatment strategy for dry AMD and STGD. Serum retinol is maintained in circulation as a tertiary complex with retinol-binding protein (RBP4) and transthyretin (TTR). Without interacting with TTR, the RBP4-retinol complex is rapidly cleared due to glomerular filtration. Retinol binding to RBP4 is required for formation of the RBP4-TTR complex; apo-RBP4 does not interact with TTR. Importantly, the retinol-binding site on RBP4 is sterically proximal to the interface mediating the RBP4-TTR interaction. Without wishing to be bound by any scientific theory, the data herein show that small molecule RBP4 antagonists displacing retinol from RBP4 and disrupting the RBP4-TTR interaction will reduce serum retinol concentration, inhibit retinol uptake into the retina and act as indirect visual cycle inhibitors reducing formation of cytotoxic A2E.

Serum RBP4 as a Drug Target for Pharmacological Inhibition of the Visual Cycle

As rates of the visual cycle and A2E production in the retina depend on the influx of all-trans retinol from serum to the RPE (FIG. 4), it has been suggested that partial pharmacological down-regulation of serum retinol may represent a target area in dry AMD treatment (11). Serum retinol is bound to retinol-binding protein (RBP4) and maintained in circulation as a tertiary complex with RBP4 and transthyretin (TTR) (FIG. 5). Without interacting with TTR, the RBP4-retinol complex is rapidly cleared from circulation due to glomerular filtration. Additionally, formation of the RBP4-TTR-retinol complex is required for receptor-mediated all-trans retinol uptake from serum to the retina.

Without wishing to be bound by any scientific theory, visual cycle inhibitors may reduce the formation of toxic bisretinoids and prolong RPE and photoreceptor survival in dry AMD. Rates of the visual cycle and A2E production depend on the influx of all-trans retinol from serum to the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex in serum is required for retinol uptake from circulation to the RPE. Retinol-binding site on RBP4 is sterically proximal to the interface mediating the RBP4-TTR interaction. RBP4 antagonists that compete with serum retinol for binding to RBP4 while blocking the RBP4-TTR interaction would reduce serum retinol, slow down the visual cycle, and inhibit formation of cytotoxic bisretinoids.

RBP4 represents an attractive drug target for indirect pharmacological inhibition of the visual cycle and A2E formation. The retinol-binding site on RBP4 is sterically proximal to the interface mediating the RBP4-TTR interaction. Retinol antagonists competing with serum retinol for binding to RBP4 while blocking the RBP4-TTR interaction would reduce serum RBP4 and retinol levels which would lead to reduced uptake of retinol to the retina. The outcome would be visual cycle inhibition with subsequent reduction in the A2E synthesis.

A synthetic retinoid called fenretinide [N-(4-hydroxy-phenyl)retinamide, 4HRP] previously considered as a cancer treatment (29) was found to bind to RBP4, displace all-trans retinol from RBP4 (13), and disrupt the RBP4-TTR interaction (13,14).

Fenretinide was shown to reduce serum RBP4 and retinol (15), inhibit ocular all-trans retinol uptake and slow down the visual cycle (11). Importantly, fenretinide administration reduced A2E production in an animal model of excessive bisretinoid accumulation, Abca4−/− mice (11). Pre-clinical experiments with fenretinide validated RBP4 as a drug target for dry AMD. However, fenretinide is non-selective and toxic. Independent of its activity as an antagonist of retinol binding to RBP4, fenretinide is an extremely active inducer of apoptosis in many cell types (16-19), including the retinal pigment epithelium cells (20). It has been suggested that fenretinide's adverse effects are mediated by its action as a ligand of a nuclear receptor RAR (21-24). Additionally, similar to other retinoids, fenretinide is reported to stimulate formation of hemangiosarcomas in mice. Moreover, fenretinide is teratogenic, which makes its use problematic in Stargardt disease patients of childbearing age.

As fenretinide's safety profile may be incompatible with long-term dosing in individuals with blinding but non-life threatening conditions, identification of new classes of RBP4 antagonists is of significant importance. Compound 1, a non-retinoid RBP4 ligand, was originally identified in a screen for compounds that may improve insulin sensitivity. It was confirmed that Compound 1 displaces retinol from RBP4, disrupt retinol-induced RBP4-TTR interaction, and reduce serum REBP4 levels. In addition, it was established that Compound 1 inhibits bisretinoid accumulation in the Abca4−/− mouse model of excessive lipofuscinogenesis which justifies additional evaluation of Compound 1 and its analogues as a treatment for dry AMD and Stargardt disease.

The present invention relates to compounds of Formulas I-IV for treatment of macular degeneration and Stargardt Disease. Disclosed herein is the ophthalmic use of RBP4 antagonist compounds of Formulas I-IV. Compound 1, also a RBP4 antagonis, was originally developed as an anti-diabetic agent (12). However, its administration did not improve insulin sensitivity in mouse diabetes models. The compounds of Formulas I-IV disclosed herein behave analogously to Compound 1.

Currently, there is no FDA-approved treatment for dry AMD or Stargardt disease, which affects millions of patients. An over the counter, non FDA-approved cocktail of antioxidant vitamins and zinc (AREDS formula) is claimed to be beneficial in a subset of dry AMD patients. There are no treatments for Stargardt disease. The present invention identified non-retinoid RBP4 antagonists that are useful for the treatment of dry AMD and other conditions characterized by excessive accumulation of lipofuscin. Without wishing to be bound by any scientific theory, as accumulation of lipofuscin seems to be a direct cause of RPE and photoreceptor demise in AMD and STGD retina, the compounds described herein are disease-modifying agents since they directly address the root cause of these diseases. The present invention provides novel methods of treatment that will preserve vision in AMD and Stargardt disease patients, and patients' suffereing from conditions characterized by excessive accumulation of lipofuscin.

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1. A method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

wherein ring A is benzene optionally further substituted; R¹ is an optionally substituted branched C₃₋₆ alkyl group; X¹ is an O, S, SO, SO₂ or NH; X² is a bond or a C₁₋₃ alkylene group; ring B is azetidine, pyrrolidine or piperidine; X³ is CO or SO₂; R² is a substituent, provided that (1) when —X¹—X²— is —NH— and ring B is piperidine, then X³ is CO; (2) when X³ is CO, then R² is not a tert-butoxy group, or a salt thereof, or a pharmaceutically acceptable salt thereof.
 2. A method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

wherein ring A is a benzene ring optionally further substituted; ring B is a piperazine ring optionally further substituted; and R is a substitutent, or a pharmaceutically acceptable salt thereof.
 3. A method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

wherein A is O, NH, or S; B is a bond, —(C₂-C₇)alkyl, —(C₂-C₇)alkenyl, —(C₃-C₈)cycloalkyl, —(C₂-C₇) heteroalkyl, —(C₃-C₈) heterocycloalkyl, —(C₃-C₈)cycloalkenyl, —(C₃-C₈)heterocycloalkenyl; D is isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, sec-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl, methylenecyclopropyl, methylenecyclobutyl, methylenecyclopentyl; E is (C═O)—OR, —O—(C═O)—R, —(C═O)—R, —OR, a carboxylic acid bioisostere, —(C═O)—NR¹R, NR¹—(C═O)—R, —(C₁-C₇)alkyl-(C═O)—OR, or —(C₁-C₇)alkyl-(C═O)NR¹R;

R is H or G is OR¹, —(C₁-C₆)alkyl, —(C₁-C₆)alkyl-OR¹, halogen, —CO₂R¹, —(C₁-C₆)alkyl-CO₂R¹, NHR¹, —(C₁-C₆)alkyl-NHR¹, —(C═O) NHR¹, —(C₁-C₆)alkyl-(C═O)NHR¹, —NHR¹(C═O)R¹, —(C₁-C₆)alkyl-NHR¹(C═O)R¹; R¹ is H or —(C₁-C₆)alkyl; X is a halogen; or an active metabolite, or a pharmaceutically acceptable prodrug, salt, or solvate thereof.
 4. A method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith, comprising administering to the mammal an effective amount of a compound having the structure:

wherein ring A is a 5-membered non-aromatic heterocycle optionally further substituted by one substitutent; ring B is an optionally further substituted benzene ring; and X is a bond, O, CH₂O, OCH₂, CH₂, (CH₂)₂, S, CH₂S, SCH₂, S(O), CH₂S(O), S(O)CH₂, S(O)₂, CH₂S(O)₂OR S(O)₂CH₂, provided that {(3S,5R)-1-[4-(trifluoromethyl)benzyl]-5-[4-(trifluoromethyl)phenyl]pyrro-lidin-3-yl}acetic acid, {(3S,5R)-1-[2,5-bis(trifluoromethyl)benzyl]-5-[4-(trifluoromethyl)phenyl]-pyrrolidin-3-yl}acetic acid, {4-oxo-3-[(3-(trifluoromethyl)phenyl]-1,3-thiazolidin-5-yl}acetic acid, {2-oxo-1-[3-(trifluoromethyl)phenyl]pyrrolidin-3-yl}acetic acid, {3-[4-fluoro-3-(trifluoromethyl)phenyl]-4-oxo-1,3-oxazolidin-5-yl}acetic acid, {4-oxo-3-[3-(trifluoromethyl)phenyl]-1,3-oxazolidin-5-yl}acetic acid, {3-[2-chloro-5-(trifluoromethyl)phenyl]-4-oxo-1,3-thiazolidin-5-yl}acetic acid, and {5-oxo-1-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1H-pyrazol-3-yl}acetic acid are excluded, or a pharmaceutically acceptable salt thereof.
 5. The method of claim 1, wherein the disease is further characterized by bisretinoid-mediated macular degeneration.
 6. The method of claim 1, wherein the amount of the compound is effective to lower the serum concentration of RBP4 in the mammal or lower the retinal concentration of a bisretinoid in lipofuscin in the mammal.
 7. (canceled)
 8. The method of claim 5, wherein the bisretinoid is A2E, isoA2E, A2-DHP-PE or atRAL di-PE.
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. The method of claim 1, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration, dry (atrophic) Age-Related Macular Degeneration, Stargardt Disease, Best disease, adult vitelliform maculopathy or Stargardt-like macular dystrophy.
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. The method of claim 2, wherein the disease is further characterized by bisretinoid-mediated macular degeneration.
 19. The method of claim 2, wherein the amount of the compound is effective to lower the serum concentration of RBP4 in the mammal or lower the retinal concentration of a bisretinoid in lipofuscin in the mammal.
 20. The method of claim 18, wherein the bisretinoid is A2E, isoA2E, A2-DHP-PE or atRAL di-PE.
 21. The method of claim 2, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration, dry (atrophic) Age-Related Macular Degeneration, Stargardt Disease, Best disease, adult vitelliform maculopathy or Stargardt-like macular dystrophy.
 22. The method of claim 3, wherein the disease is further characterized by bisretinoid-mediated macular degeneration.
 23. The method of claim 3, wherein the amount of the compound is effective to lower the serum concentration of RBP4 in the mammal or lower the retinal concentration of a bisretinoid in lipofuscin in the mammal.
 24. The method of claim 22, wherein the bisretinoid is A2E, isoA2E, A2-DHP-PE or atRAL di-PE.
 25. The method of claim 3, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration, dry (atrophic) Age-Related Macular Degeneration, Stargardt Disease, Best disease, adult vitelliform maculopathy or Stargardt-like macular dystrophy.
 26. The method of claim 4, wherein the disease is further characterized by bisretinoid-mediated macular degeneration.
 27. The method of claim 4, wherein the amount of the compound is effective to lower the serum concentration of RBP4 in the mammal or lower the retinal concentration of a bisretinoid in lipofuscin in the mammal.
 28. The method of claim 26, wherein the bisretinoid is A2E, isoA2E, A2-DHP-PE or atRAL di-PE.
 29. The method of claim 4, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration, dry (atrophic) Age-Related Macular Degeneration, Stargardt Disease, Best disease, adult vitelliform maculopathy or Stargardt-like macular dystrophy. 